Electrophoresis in a suitable carrier, e.g. in agarose, polyacrylamide or starch gel, or alternatively in filter paper or acetate foil is used for separating protein constituents of biological fluids. It is also known that the separation can be considerably improved if following on the electrophoretic preliminary separation in a carrier, immunomigration is subsequently performed at right angles to the first separating direction. This process is called crossed-electrophoresis.
Laurell disc electrophoresis described in Hoppe-Seyler's Z. Physiol. Chem., vol. 354, pp. 673 - 681 (1973) uses a base plate with a well into which the antiserum-containing agarose solution is poured. In the first phase of this process a polyacrylamide gel poured into capillary tubes 5 cm long and with an internal diameter of 1 mm is subjected to micro-disc electrophoresis. These polyacrylamide gel cylinders are pressed into a slit previously cut in the antiserum-containing agarose layer close to one edge of the well. The pressing of the polyacrylamide cylinder into the agarose carrier merely produces a mechanical connection between the two carrier layers. As a result of this solely mechanical contact between the two carriers, the transition of the protein fractions from the first gel to the second is made more difficult.
If the same carrier, e.g. the same gel was used for both crossed-electrophoresis phases, many technical difficulties would admittedly be obviated, but protein separation is not optimum. Furthermore, through the use of different gels, it is possible to combine their individual advantages but then considerable technical problems occur resulting from the fact that the second gel has to be poured onto the first gel to permit the transition of the protein fractions into the second gel. Due to the differing endosmosis in different gels, a waterflow occurs at the interface which impairs or completely prevents the further migration of antigens. Due to the varying drying characteristics of different gels (e.g. agarose gels dry out whereas polyacrylamide gels shrink) a separation of the gels can easily occur at the interface. In addition, different gels have different optimum layer thicknesses which also makes it more difficult to combine different gels.
The problem of the present invention is to develop a suitable chamber for performing the second crossed-eletrophoresis phase preferably using different carriers or gels for the electrophoresis step and the electroimmunomigration step, in order in this way to utilise the advantages of different gels during separation. In addition, a process for performing the electroimmunomigration step using the novel chamber is proposed.
Therefore the object of the invention is a chamber for the second crossed-electrophoresis step following on the prior electrophoretic separation of a protein-containing sample in a first carrier, accompanied by separation in a second carrier in a direction at right angles to the first separating direction, which comprises a base plate with a well for a second carrier, wherein the well in the base plate is subdivided into a migration well and a connecting well by a depot for receiving the first carrier, whereby a lid is provided for the depot which in each case provides a vertically variable slit, via which the first carrier in the depot is in contact with the second carrier in the wells.
This chamber, which will be described in greater detail hereinafter, makes it possible for the first time to satisfactorily perform electroimmunomigration in a technically uncomplicated manner in a carrier differing from that used for the previous electrophoresis. For example, a protein-containing biological sample can firstly be separated by means of conventional disc-electrophoresis or by means of flat disc-electrophoresis using a polyacrylamide gel. The particular advantage of a polyacrylamide gel is its concentration effect so that even small quantities of low protein fluids can be investigated, e.g. body fluids of small test animals (rats, mice, chickens, etc.) or the cerebrospinal fluid of humans. Thus it is possible to work with very small quantities of test fluid which obviates any prior concentration which can easily lead to denaturing of certain antigens.
The second phase, i.e. electroimmunomigration, can be performed using the chamber according to the invention in a different gel, e.g. in conventional agarose gel, wherein much better defined precipitates are obtained than in polyacrylamide gel. The optimum layer thickness for agarose gel is admittedly about 1 mm, but in the novel chamber this layer can without difficulty be poured onto the polyacrylamide gel, although the layer thickness of the latter is generally a few millimetres, preferably approximately 3 mm. It has surprisingly been found that the vertically variable slit by means of which the two different gels are in contact is adequate, and it has been found that substantially all the antigens pass from the first gel into the second gel. Thus, improved protein analysis is possible using extremely small quantities of test fluid with low antigen concentration leading to a representation of the antigens in the form of well-defined precipitate peaks.
It is often more advantageous to use cellulose-acetate membrane foils in place of the polyacrylamide gel as the first carrier. They have proved particularly suitable for protein electrophoresis and are at present the most frequently used carrier material for this purpose in clinical chemical laboratories. Cellulose-acetate foils are also a very suitable carrier medium for immunodiffusion methods and immunoelectrophoresis. Agarose is best suited as the carrier material for the second phase of crossed-immunoelectrophoresis with its various modifications, whereas for the second phase of crossed-immunoelectrophoresis cellulose-acetate membrane foil can without difficulty be used as the carrier material. In the second phase considerable difficulties occur because evaporation leads to drying or shrinkage of the foil. It is substantially impossible to work with an intermediate layer.
In the chamber according to the invention these difficulties can be obviated in that the depot lid has holes in the area adjacent to the migration well. Warm agarose can be poured through one of these holes onto the cellulose-acetate strips in the closed depot obtained in the first crossed-electrophoresis step, so that the cellulose acetate membrane foil is embedded in agarose and evaporation with its consequences is prevented.
According to a further embodiment of the chamber of the invention, a plurality of individual chambers is combined to form a multiple chamber. This permits the simultaneous performance of the second crossed-electrophoresis step with a plurality of carriers used in the first separating step.